The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) protects plasmid pUC-18 DNA against OH-mediated strand breaks

doi:10.1016/S0891-5849(98)00117-8

Free Radical Biology and Medicine

Volume 25, Issue 8, 15 November 1998, Pages 875-880

https://doi.org/10.1016/S0891-5849(98)00117-8 Get rights and content

Abstract

Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373–378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by radical dot OH radicals generated from Fe(II) plus H₂O₂ or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20–30 μM) were able to half-protect DNA from iron (1–7.5 μM)-mediated radical dot OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit radical dot OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze Fenton reactions.

Introduction

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) [1], [2], [3] has been used for iron metabolism studies and examined for its potential use in the treatment of secondary iron overload pathologies [1], [2], [3], [4], [5] that occur in iron-loading anemias such as β-thalassemias, porphyria cutanea tarda and alcoholic cirrhosis [6]. Experimental and oral administration of PIH induces iron excretion in rats and humans resulting in negative iron balance without serious side effects [4], [7], [8], [9], [10], [11], [12]. PIH-induced iron excretion in rats occurs mainly through bile [5], [12] and is approximately equivalent to that obtained with a comparable dose of desferrioxamine (DFO) given parenterally [7].

Induction of iron excretion can decrease hepatic oxidative stress in iron overload (reviewed in ref. 13). It is well known that iron catalyzes the formation of highly reactive hydroxyl radicals ( radical dot OH) that can mediate injury to various biomolecules [14]. Iron-mediated lipid peroxidation, depletion of low-molecular weight antioxidants and single- and double-strand breaks in DNA have been implicated in the pathophysiology of iron overload diseases [13], [15], [16], [17], [18]. In humans, iron overload correlates with DNA alterations and cancer [13], [19], [20]. It was reported that the risk of hepatocellular carcinoma is greatly increased in iron overload patients [21], [22], [23]. Moreover, i.p. injections of ferric-NTA to experimental animals promotes renal proximal tubular necrosis and a high incidence (60–92%) of renal adenocarcinoma [17], [24].

We recently demonstrated that PIH and some of its analogs have antioxidant activity against iron-mediated lipid peroxidation of liposomes [25] and can prevent radical dot OH-mediated (formed via Fe(III)EDTA plus ascorbate) release of TBARS from 2-deoxyribose as well as the release of ethylene from 2-keto-4-methiobutyric acid [25]. More recently PIH was shown to inhibit peroxidation and retinal electrophysiological alterations secondary to asphyxia-reoxygenation-induced oxidative stress to newborn animals [26]. Here, we report on the ability of PIH and two of its analogues (pyridoxal benzoyl hydrazone, PBH, and salicylaldehyde isonicotinoyl hydrazone, SIH; Fig. 1) to prevent plasmid pUC-18 DNA strand breaks induced by radical dot OH radicals.

Section snippets

Chemicals and solutions

PIH, SIH and PBH were synthesized as previously described [25], [27]. All solutions were prepared in Milli-Q quality water. Stock solutions (0.75 mM) of the iron chelators were prepared daily in 1 mM Hepes buffer pH 7. Stock solutions of Fe(II) were freshly prepared (1 mM) in water previously bubbled with nitrogen. Dilutions of Fe(II) stock solutions were done in deoxygenated water to prevent autoxidation. Escherichia coli cells were transformed with pUC-18 plasmid DNA (2,686 base-pairs) and

Characterization of the damage to plasmid pUC-18

Control preparations of pUC-18 contained 80 ± 3.9% supercoiled (SC) DNA (Fig. 2, lane A), levels of which are not affected by incubation with 0.3 mM H₂O₂ for 5–30 min. However, addition of increasing concentrations of Fe(II) causes, after 5 min, breakage of pUC-18 (Fig. 2, lanes B–G). With 1 μM Fe(II) there was breakage of approximately half the SC DNA while its complete elimination occured with 15 μM Fe(II), in the presence of 0.3 mM H₂O₂ (Fig. 2, lanes B–G).

The breakage of pUC-18 is also

Conclusions

Free radical damage to DNA has been implicated in the development of renal and hepatic cancer in iron overload [13], [21], [22], [23], [24] and, in general, there is thought to be a link between free radical damage to DNA and cancer [15], [19], [20]. Therefore, studying iron-mediated in vitro damage to DNA is relevant for understanding the biochemical basis of the proposed iron-mediated carcinogenesis [19], [20].

In the present paper we demonstrate that the iron chelators, PIH, SIH and PBH,

Acknowledgements

This work was supported by grants from MRC (Canada) to Drs. P. Ponka and H.M. Schulman. Dr. M. Hermes-Lima was supported by grants from CNPq, PADCT, PRONEX-97 and FAP-DF (Brazil) as well as by an award from the Lady Davis Institute (Canada). The authors thank Drs. M. Andrews and J. Yan (Department of Chemistry, McGill University) for preliminary EPR studies and Alice Mota (Universidade de Brasilia) for general assistance. This research is dedicated to the memory of Professor G. Cilento

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Original ContributionsThe iron chelator pyridoxal isonicotinoyl hydrazone (PIH) protects plasmid pUC-18 DNA against OH-mediated strand breaks

Abstract

Introduction

Section snippets

Chemicals and solutions

Characterization of the damage to plasmid pUC-18

Conclusions

Acknowledgements

FEBS Lett.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Blood

J. Lab. Clin. Med.

Meth. Enzymol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Gastroenterology

Free Radic. Biol. Med.

Chem. Biol. Interact.

J Free Radic. Biol. Med.

Free Radic. Biol. Med.

Arch. Biochem. Biophys.

Inorg. Chim. Acta

Physiology and pathophysiology of iron metabolismImplications for iron chelation therapy in iron overload

Secondary iron overload

Original Contributions
The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) protects plasmid pUC-18 DNA against OH-mediated strand breaks